In U.S. Pat. No. 4,666,836, issued May 19, 1987 to M. Inouye and K. Nakamura, entitled "Novel Cloning Vehicles For Polypeptide Expression In Microbial Hosts," a class of recombinant bacterial plasmid cloning vehicles for expression of exogenous genes in transformed bacterial hosts is disclosed, comprising a DNA insert fragment coding for the desired polypeptide, linked in reading phase with one or more functional fragments derived from an outer membrane protein gene of any Gram-negative bacterium. In a preferred embodiment, the exogenous DNA codes for mammalian hormones, enzymes or immunogenic proteins (or intermediates therefor), the functional fragments are derived from the lipoprotein gene of E. coli, and the desired polypeptide is expressed in E. coli transformants preferred embodiment, the DNA sequence coding ired protein is linked with and is expressed in with four specific functional fragments associated with the E. coli lipoprotein gene, namely, the promoter, the 5'-untranslated region, the 3'-untranslated region and the transcription termination site of that gene.
These expression plasmids may also include a second promoter, preferably an inducible promoter and most preferably the E. coli .beta.-galactosidase or "lac" promoter, which is inserted immediately downstream of the lipoprotein promoter so that the exogenous DNA is expressed only in the presence of a "lactose inducer." When induced, the DNA coding for the desired polypeptide is transcribed from both promoters, thereby increasing the yield of the desired product. Accordingly, both constitutive and inducible gene expression may be achieved using the cloning vehicles of the invention of U.S. Pat. No. 4,666,836.
However, it is disclosed in U.S. Pat. No. 4,666,836, that with the inducible cloning vehicles, special E. coli strains are preferred for use as transformants, specifically, those which can overproduce the lactose repressor molecule. In the wild-type E. coli cell, only about 10 copies of the lactose repressor molecule are maintained in the cell at any one time, which is just enough to repress (i.e., inhibit the expression of) the one lacZ gene normally contained in the cell. This is insufficient, however, to block the expression of the exogenous DNA cloned in an inducible expression plasmid of the invention of U.S. Pat. No. 4,666,836, since 10 to 20 copies of the cloning vehicle, each containing an active lac promoter, may exist in each cell at a given time. Therefore, much larger amounts of the lactose repressor are required, and for this purpose, the strain used for transformation is preferably a special E. coli strain JA221/F' lac.sup.Iq lac.sup.+ pro.sup.+, which carries the mutant lac.sup.Iq gene. The lac.sup.Iq gene is a mutant of lacI, the "normal" gene coding for the lactose repressor. The mutant gene overproduces the lactose repressor, providing about 100-150 molecules/cell at any given time. The lac.sup.Iq gene is carried on the plasmid F-prime in this E. coli strain.
The fact that this scheme necessitates expression of the desired polypeptide in transformants carrying the plasmid F-prime gives rise to certain disadvantages. First of all, the class of recipients for the inducible expression plasmids of U.S. Pat. No. 4,666,836 is inherently limited to those E. coli strains which carry the lac.sup.I q gene, since strains which lack this gene would not produce enough of the lactose repressor and would therefore continuously generate the desired expression product.
Secondly, the F-prime plasmid is a sex factor which causes E. coli cells to conjugate, resulting in transfer of the F-prime plasmid from one cell to another. The use of E. coli strains carrying this factor for eukaryotic gene cloning is complicated, thereby reducing still further the applicability of the scheme on which U.S. Pat. No. 4,666,836 is based.
Finally, since there are usually 2 or 3 copies of the F-prime plasmid in a cell (each of which maintains about 100-150 lactose repressor molecules), and since each cell also contains 10-20 copies of one of the inducible expression plasmids of U.S. Pat. No. 4,666,836 (each carrying a functional lac promoter), the ratio of repressor molecules to lac promoters will vary widely from cell to cell, and in some instances will not achieve complete repression of the desired expression product.
It is therefore the principal object of the present invention to provide a new class of plasmid cloning vehicles with which these disadvantages may be overcome.